Novel lactobacillus plantarum and composition comprising the same

ABSTRACT

Lactobacillus plantarum CJLP243 (KCTC 11045P), a composition for treating intestinal diseases comprising Lactobacillus plantarum CJLP243, and a composition for enhancing immune response comprising Lactobacillus plantarum CJLP243.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of U.S. application Ser. No.13/503,800, filed on Apr. 24, 2012, which is a 35 U.S.C. § 371 NationalStage Application of International Application No. PCT/KR2010/007431,filed on Oct. 27, 2010, which claims priority under 35 U.S.C. § 119(a)to Korean Application No. 10-2009-0102822, filed on Oct. 28, 2009, eachof which are incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to novel Lactobacillus plantarum and acomposition comprising the same, and more particularly, to novelLactobacillus plantarum for preventing or treating intestinal diseasesand immune diseases and a composition comprising the same.

BACKGROUND ART

Lactic acid bacteria found in Korean traditional fermented food, e.g.,kimchi, inhabit the digestive system of a human body and decomposefibrous materials and complex proteins into important nutrients. Viablemicroorganisms like lactic acid bacteria are beneficial togastrointestinal tracts of hosts such as animals including humans due tothem improving intestinal microorganism environments of the hosts andare referred to as probiotics. Probiotics are required to have excellentacid resistance and bile resistance, and strong adhesion to intestinalepithelial cells since they need to be orally administered, reach thesmall intestine and adhere to the surface of the small intestine inorder to be effective as probiotics.

Lactobacillus sp. lactic acid bacteria are probiotics widely found inKorean traditional fermented food such as Kimchi. Lactobacillus sp.microorganisms produce lactic acid under homo-fermentation orhetero-fermentation and are widely found in intestinal tracts of animalsincluding humans, and in fermentation of dairy products and vegetables.Lactobacillus sp. microorganisms maintain an acidic pH balance tosuppress propagation of hazardous bacteria such as E. coli orClostridium and relieve diarrhea and constipation. Lactobacillus sp.microorganisms also synthesize vitamins, have anti-cancer activities,and reduce serum cholesterol. Acidophillin produced by lactobacillus isknown to inhibit the growth of shigella, salmonella, staphylococcus, E.coli, or the like. Acidophillin also suppresses the propagation ofcausative microorganisms of diarrhea and normalizes intestinal flora toprevent diarrhea (Michael and Philippe, Probiotics and prebiotics:Effects on diarrhea, The journal of nutrition, Volume 137, March 2007,pages 803S-811S; Roberfroid, Prebiotics and probiotics: Are theyfunctional foods?, American journal of clinical nutrition, Volume 71,June 2000, pages 1682S-1687S).

Based on the Lactobacillus sp. microorganism's functions as statedabove, research into Lactobacillus sp. microorganism as probiotics andlivestock feed is being vigorously conducted. Bacterial diarrhea inlivestock causes mortality and a reduction in the rate of weight gain.Thus, a method of adding antibiotics to livestock feed has been widelyused in order to prevent bacterial diarrhea and increase theproductivity of livestock. However, due to the appearance of antibioticresistant bacteria and remaining antibiotics in the livestock, the useof antibiotics has been limited and organic methods of raising livestockhave been recommended (Korean Patent Publication No. 1998-78358)(McEwenand Fedorka-Cray, Antimicrobial use and resistance in animals, Clinicalinfectious Diseases, Volume 34, June 2002, pages S93-S106).

In addition, lactic acid bacteria such as Lactobacillus sp.microorganism are known to have immune response-enhancing effects. Assuch, research on the mechanism of effects of lactic acid bacteria onimmune response-enhancing effects has been conducted. Even thoughspecific mechanisms have not yet been revealed, it is known that lacticacid bacteria are orally administered and inhabit the intestines toinfluence the intestinal immune system. For example, the intake oflactic acid bacteria via yogurt is known to increase antibioticactivities of lymphocytes of Peyer's patches. Lactic acid bacteria areknown to enhance IgA response according to research into animals andhumans. In addition, the immune system representing resistance in thebody against external microbial pathogens is divided into innateimmunity and adaptive immunity, both of which are influenced by lacticacid bacteria. According to the innate immunity of the intestinal immunesystem, the lactic acid bacteria are known to prevent and kill pathogensthereby having a function of maintaining healthy conditions againstinfection. Innate immune reaction plays an important role in suppressingthe increase of the number of external pathogens at an early stage of aninfection. Additionally, innate immune reaction delivers antigens andco-stimulatory molecules, thereby inducing the activation of theadaptive immunity subsequent to the innate immune system. Representativeimmunocytes related to the innate immune reaction includes NK cells,neutrophiles, macrophages, and dendritic cells (Fearon D T, Locksley RM, Science 1996, 272:50-53, The instructive role of innate immunity inthe acquired immune response). Adaptive immunity substantially inducesthe removal of external pathogen when a subject is infected, and theimmunocytes corresponding to adaptive immunity include T lymphocytes andB lymphocytes. Accordingly, physical resistance to external pathogensmay depend on the enhancement of the activity of adaptive immunity(Gowans J L., Immunology Today. 1996 Jun.;17(6):288-91, The lymphocyte—adisgraceful gap in medical knowledge).

According to the adaptive immunity, macrophages that decompose antigensto bring them into contact with T lymphocytes are activated to increasethe production of a variety of cytokines, particularly, interleukin,IL-12, and IL-18. In this regard, some components of the cell walls oflactic acid bacteria activate NF-κB and STAT signal transfers in themacrophages to increase the production of the cytokines. In addition, itis known that lactic acid bacteria increase the production of IL-12,IL-18, and TNF-α in antigens presenting cells such as dendritic cellsoften found in lymphatic nodes and mucosa of the digestive system.Furthermore, lactic acid bacteria are known to increase the expressionof a surface molecule in dendritic cells that activate T lymphocytessuch as MHC class II and B7-2 (Cross et. al.,Anti-allergy properties offermented foods: an important immunoregulatory mechanism of lactic acidbacteria?, International Immunopharmacology, Volume 1, May 2001, pages891-901).

Much research on the interrelation between Lactobacillus sp.microorganisms and immune reactions has been carried out. Specifically,some Lactobacillus Lactobacillus sp. (e.g. L. fermentum) is known toenhance an antigen-specific immune reaction, and thus its use as anadjuvant in bacteria (e.g. diphtheria, tetanus) or virus (e.g.influenza, polio) vaccines was tried (de Vrese et al., 2005; Olivares etal., 2007; West et al., 2008). Immune-enhancing effects of LactobacillusLactobacillus sp. microorganism are considered to result from theenhanced activity of Th1-type cytokine-producing T lymphocyte byspecific Lactobacillus Lactobacillus sp. microorganisms, whicheffectively induce the growth of general immune cells and the activityof T cells or B cells in adaptive immunity (Mohamadzadeh et al.,Lactobacilli activate human dendritic cells that skew T cells toward Thelper 1 polarization., Proc Natl Acad Sci USA. 2005 22;102(8):2880-2885). In order to measure the enhanced activity of Tlymphocyte, the study of measuring the amount of produced IFN-γ isactively performed in recent years (Shida et al., 2006; Foligne et al.,2007). It is known that the induction of general immune cell growth mayprevent or treat digestive tract (intestinal) infection (Jain S, YadavH, Sinha P R. Probiotic dahi containing Lactobacillus caseiLactobacillus casei protects against Salmonella enteritidis infectionand modulates immune response in mice. J Med Food. 2009June;12(3):576-83.), genitourinary infection (Zarate G, Santos V,Nader-Macias ME. Protective effect of vaginal Lactobacillus paracaseiLactobacillus paracasei CRL 1289 against urogenital infection producedby Staphylococcus aureus in a mouse animal model. Infect Dis ObstetGynecol. 2009;2009:48358. Epub 2007 Mar. 29.), respiratory infection(Yasuda Y, Matsumura Y, Kasahara K, Ouji N, Sugiura S, Mikasa K, Kita E.Microbial exposure early in life regulates airway inflammation in miceafter infection with Streptococcus pneumoniae with enhancement of localresistance. Am J Physiol Lung Cell Mol Physiol. 2009 Sep. 25 [Epub aheadof print]), helicobacter infection (Boyanova L, Stephanova-KondratenkoM, Mitov I. Anti-Helicobacter pylori activity of Lactobacillusdelbrueckii subsp. bulgaricus strains: preliminary report. Lett ApplMicrobiol. 2009 May;48(5):579-84. Epub 2009 Mar. 9.), and allergicreactions (Ouwehand A C, Nermes M, Collado M C, Rautonen N, Salminen S,Isolauri E. Specific probiotics alleviate allergic rhinitis during thebirch pollen season. World J Gastroenterol. 2009 Jul. 14;15(26):3261-8).

T lymphocytes control adaptive immunity which may be classified into aTh1 response as cellular immunity and a Th2 response as humoralimmunity. Th1 and Th2 responses produce different cytokines in theantigen presenting cells. In the Th1 response, the production of IL-2,IL-12, IL-18, interferon-γ(IFN-γ is dominant. In the Th2 response, theproduction of PGE2, IL-4, and IL-10 is dominant. The Th1 and Th2responses are required to be balanced. If the Th1 and Th2 responses arenot balanced, a variety of immune diseases occur. Th1 cells mainly fightwith pathogens, but Th2 cells are mainly related to allergies andinflammatory responses. When the Th1 and Th2 responses are in normalconditions, Th2 cells protect the human body from dust and undesiredmaterials. However, if the Th2 cells excessively respond, the productionof IgE antibodies increases, thereby causing allergic responses toproteins that are not hazardous to the human body, such as pollen andfood. Thus, the ratio of Th1 and Th2 responses should be balanced.Excessive or insufficient response of one of them causes diseases. Inaddition, continuous secretion of cortisol due to continuous stressdecreases Th1 response and increases Th2 response, thereby causingcancer, atopic diseases, allergies, and autoimmune diseases (Elenkov andChrousos, Stress hormones, Th1/Th2 patterns, pro/anti-inflammatorycytokines and susceptibility to disease, Trends in Endocrinology andMetabolism, Volume 10, November 1999, pages 359-368).

According to in vivo experiments, lactic acid bacteria increase theproduction of IL-2 and IFN-γ that are Th1 cytokines in T lymphocytes andsuppress the production of IL-4 and IL-5 that are Th2 cytokines(Matsuzaki et. al., The effect of oral feeding of Lactobacillus caseistrain Shirota on immunoglobulin E production in mice, Journal of DairyScience, Volume 81, January 1998, pages 48-53). Meanwhile, IL-12 andIL-18 are important cytokines to differentiate Th0 lymphocytes into Th1lymphocytes and produced in macrophages or dendritic cells. It is knownthat the production of IL-12, IL-18 and IFN-α increases depending uponthe concentration of lactic acid bacteria, when splenocytes ormacrophages are treated with lactic acid bacteria during the culturing.As such, lactic acid bacteria increase the production of IL-12, IL-18and IFN-α in macrophages, thereby promoting the differentiation of Th0into Th1 and inducing the formation of IFN-γ and thus lactic acidbacteria play a role in balancing the Th1/Th2 in a Th2-response primedstate (Cross et. al., Anti-allergy properties of fermented foods: animportant immunoregulatory mechanism of lactic acid bacteria?,International Immunopharmacology, Volume 1, May 2001, pages 891-901).Thus, lactic acid bacteria are known to prevent and treat cancers,atopic diseases, allergies, and autoimmune diseases induced by theTh1/Th2 imbalance caused by the over-response of Th2.

DISCLOSURE OF INVENTION Technical Problem

While searching for novel lactic acid bacteria having excellentimmune-enhancing activities in comparison with the conventional lacticacid bacteria, the present inventors isolated and identifiedLactobacillus sp. bacteria isolated from Korean traditional fermentedfood.

Thus, the present invention provides Lactobacillus sp. bacteria strainshaving excellent effects on enhancing immune response, particularly,excellent activities for promoting the production of IFN-γ, a Th1-typecytokine, inducing the proliferation of general immune cells, andcountering Th1/Th2 imbalance caused by an over-response of Th2, as wellas having excellent acid and bile resistance and strong adhesion tointestinal epithelial cells constituting basic properties of probiotics.

The present invention also provides a composition for preventing ortreating intestinal diseases, comprising Lactobacillus sp. bacteriastrains.

The present invention also provides a composition for enhancing immuneresponse, comprising Lactobacillus sp. bacteria strains.

Solution to Problem

According to an aspect of the present invention, there is providedLactobacillus plantarum CJLP243 (deposited in Korean Culture Center ofMicroorganisms (KCCM), Deposition date: Oct. 14, 2009, Deposition No.:KCCM11045P).

According to another aspect of the present invention, there is provideda composition for preventing or treating intestinal diseases, comprisingLactobacillus plantarum CJLP243.

According to an aspect of the present invention, there is provided acomposition for enhancing immune response, comprising Lactobacillusplantarum CJLP243.

Hereinafter, the present invention will be described more fully withreference to the accompanying drawings, in which exemplary embodimentsof the invention are shown.

Lactobacillus plantarum CJLP243 according to an aspect of the presentinvention is a novel strain of Lactobacillus plantarum isolated andidentified from Korean traditional fermented food. The Koreantraditional fermented food may be kimchi, fermented vegetables, soybeanpaste, soy sauce, chungkookjang, fermented fish, or the like, but is notlimited thereto.

A result of a 16S rRNA base sequence assay for identifying andclassifying Lactobacillus plantarum showed that Lactobacillus plantarumCJLP243 had the highest homology (99.9%) with a Lactobacillus plantarumstandard strain (Lactobacillus plantarum NBRC15891^(T), GenBankaccession number AB326351) and showed the closest molecular phylogeneticrelationship with Lactobacillus plantarum. Thus, the said microorganismwas identified as Lactobacillus plantarum, named Lactobacillus plantarumCJLP243, and deposited in the Korean Culture Center of Microorganisms(KCCM) on Oct. 14, 2009 (Deposition No.: KCCM11045P). The base sequenceof a 16S rRNA gene of Lactobacillus plantarum CJLP243 is shown at SEQ IDNO: 1 in the sequence list attached hereto.

Lactobacillus plantarum CJLP243 is a gram-positive bacterium and afacultative anaerobe that may grow in both aerobic and anaerobicconditions, does not produce spores, does not have motility, and has arod shape. Specific morphological and physiological properties ofLactobacillus plantarum CJLP243 have been analyzed according toconventional methods known in the art and are shown in Table 1 below.

TABLE 1 Morphological, physiological, and biochemical properties ResultsMorphology Rod Motility − Spore − Catalase − facultative heterofermentation Homo-hetero fermentation Proliferation at 10° C. +Proliferation at 42° C. + Proliferation at 7% NaCl + Proliferation at10% NaCl − Proliferation at pH 3.8 + Proliferation in anaerobicconditions + Production of CO₂ using glucose − Sugar-fermentationGlycerol − Erythritol − D-arabinose − L-arabinose + Ribose + D-xylose −L-xylose − Adonitol − xyloside − Galactose + D-glucose + D-fructose +D-mannose + L-sorbose − Rhamnose + Dulcitol − Inositol − Mannitol +Sorbitol + D-mannoside + D-glucoside + Glucosamine + Amygdalin +Alutin + Esculin + Salicin + Cellobiose + Maltose + Lactose +Melibiose + Sacarose + Trehalose + Inulin + Melizitose + D-raffinose +Amidon − Glycogen − Xylitol − Gentiobiose − D-turanose − D-lyxose −D-tagatose − D-fucose − L-fucose − D-arabitol − L-arabitol − Gluconate −2-gluconate − 5-gluconate − +: positive −: negative

In order to stably preserve Lactobacillus plantarum CJLP243 for a longperiod of time, the strains may be preserved by dispensing them in apreservation solution prepared by mixing water and glycerol and storingthe dispension at −70° C., or by suspending them in sterilized 10% skimmilk and lyophilizing the suspension.

In addition, Lactobacillus plantarum CJLP243 is a probiotic that iseffective for relieving intestinal disorders and enhancing immuneresponse that are general effects of lactic acid bacteria.

In this regard, a probiotic is a viable microorganism beneficial togastrointestinal tracts of hosts such as animals including humans byimproving intestinal environments. Probiotics, which are livingmicroorganisms having probiotic activities in a single or complexstrain, are beneficial to intestinal flora when administered to humansor animals in a dried or fermented form. The probiotic microorganismshould not be influenced by gastric juice and bile, and is required tobe viable in the intestine after passing through the stomach, inhabitsthe intestine, and is beneficial to intestinal flora of a host. Thus,the probiotic microorganism needs to have excellent acid resistance andbile resistance, and strong adhesion to intestinal epithelial cells. Inaddition, the probiotic microorganism is required to be stable. In thisregard, a gelatin liquefaction test, a phenylalanine deaminase test, anammonia formation test, a hemolysis test, or the like are conducted.Lactobacillus plantarum CJLP243 according to the present embodiment isnegative for the gelatin liquefaction test, the phenylalanine deaminasetest, and the ammonia formation test, and shows α-hemolysis indicatingthat Lactobacillus plantarum CJLP243 is not a pathogen (refer to Example4), has excellent acid resistance and bile resistance, and shows strongadhesion to intestinal epithelial cells (refer to Examples 2 and 3).

Lactobacillus plantarum CJLP243 may have excellent effects on relievingintestinal disorders due to excellent acid resistance and bileresistance, and shows strong adhesion to intestinal epithelial cells.

Thus, another aspect of the present invention provides a composition forpreventing or treating intestinal diseases, comprising Lactobacillusplantarum CJLP243.

The composition for treatment of intestinal diseases comprisingLactobacillus plantarum CJLP243 may be applied to prevent or treatintestinal diseases of mammals including humans, for example, livestockincluding cows, horses, and pigs. Such intestinal diseases includeintestinal infections by bacteria hazardous to intestinal environmentsand inflammatory bowel diseases, for example, infectious diarrhea causedby pathogenic microorganisms (E. coli, salmonella, and clostridium),gastroenteritis, inflammatory bowel diseases, psychogenic enteritissyndrome, overgrowth of microorganisms in the small intestine, diarrhea,or the like, but is not limited thereto. Lactobacillus plantarum CJLP243contained in the composition for treating intestinal diseases may belive or dead bacteria, and preferably live bacteria. In general, thelive bacteria treat or relieve general symptoms caused by abnormalfermentation in the intestinal flora, inhabit the intestinal tracts toprevent hazardous bacteria from adhering to the intestinal tracts inhumans and animals, and produce lactic acid to lower the intestinal pH,thereby suppressing proliferation of hazardous bacteria. In addition,the administered live bacteria produce peroxides with bacteriocin tosuppress the proliferation of hazardous bacteria and help the activityof intestinal villi to absorb nutrients. In addition, live bacteria mayproduce materials assisting absorption and utilization of nutrients,improve livestock feed requirements, and produce materials neutralizingtoxic materials produced by pathogens.

The composition for preventing or treating the intestinal diseases maybe orally administered, but the method of administering the compositionis not limited thereto. The dose may vary according to types of theintestinal disease, the degree of seriousness, age, gender, andethnicity of patients, and purposes of the treatment or prevention. Ingeneral, 10 million to 100 billion bacteria may be administered to anadult.

In addition, Lactobacillus plantarum CJLP243 has excellent effects onenhancing immune response as well as effects on relieving intestinaldisorders when compared to conventional lactic acid bacteria.Lactobacillus plantarum CJLP243 is found to promote the production ofIFN-γ, Th-1 type cytokines, thereby inducing the enhancement of Th-1type immunization. Effects on enhancing immune response of Lactobacillusplantarum CJLP243 will now be described in more detail.

When immune cells isolated from a spleen of a mouse were treated withLactobacillus plantarum CJLP243, they activated the growth of Th2response-induced general immune cells (Example 5), and increased theproduction of IFN-γ, Th1-type cytokines (Example 6). The activation ofimmune cell growth and the enhancing effects of Th1-type immune reactionare found to be significantly advantageous over other conventionallactic acid bacteria such as Lactobacillus rhamnosus GG (KCTC 5033). Inaddition, when Lactobacillus plantarum CJLP243 was orally administeredto mice for 8 weeks, it was shown that the production of IFN-γ and IL-2,a Th-1 type cytokine, was increased and the growth of T cell, CD4 Tcell, and CD8 T cell were all increased. Thus, it can be said thatLactobacillus plantarum CJLP243 produces such a large quantity of IFN-γas to promote Th1-type response, thereby not only enhancing generalimmunity but also regulating the immune imbalance of Th1/Th2.

It is known that an increase in general immune cells, specifically Tcells including CD4 T cells and CD-8 T cells, enhances immunity to beeffective for preventing or treating digestive tract (intestinal)infection, urogenital infection, respiratory infection, helicobacterinfection or allergic reactions (digestive tract (intestinal) infection;Jain S, Yadav H, Sinha P R. Probiotic dahi containing Lactobacilluscasei protects against Salmonella enteritidis infection and modulatesimmune response in mice. J Med Food. 2009 June;12(3):576-83., urogenitalinfection; Zarate G, Santos V, Nader-Macias M E. Protective effect ofvaginal Lactobacillus paracasei CRL 1289 against urogenital infectionproduced by Staphylococcus aureus in a mouse animal model. Infect DisObstet Gynecol. 2009;2009: 48358. Epub 2007 Mar. 29., respiratoryinfection; Yasuda Y, Matsumura Y, Kasahara K, Ouji N, Sugiura S, MikasaK, Kita E. Microbial exposure early in life regulates airwayinflammation in mice after infection with Streptococcus pneumoniae withenhancement of local resistance. Am J Physiol Lung Cell Mol Physiol.2009 Sep. 25. [Epub ahead of print]; or helicobacter infection (BoyanovaL, Stephanova-Kondratenko M, Mitov I. Anti-Helicobacter pylori activityof Lactobacillus delbrueckii subsp. bulgaricus strains: preliminaryreport. Lett Appl Microbiol. 2009 May;48(5):579-84. Epub 2009 Mar 9.)and allergic reactions (Ouwehand A C, Nermes M, Collado M C, Rautonen N,Salminen S, Isolauri E. Specific probiotics alleviate allergic rhinitisduring the birch pollen season. World J Gastroenterol. 2009 Jul.14;15(26):3261-8). Therefore, the experimental results show thatLactobacillus plantarum CJLP243 is effective for preventing or treatingdigestive tract (intestinal) infection, urogenital infection,respiratory infection, helicobacter infection or allergic reaction, dueto the enhanced general immunity resulting from the activation of T cellgrowth.

Recently, it has been reported that Th2 cells relatively increase inperipheral blood and skin lesions in patients having atopic dermatitis(Miraglia et. al, Immune dysregulation in atopic dermatitis, Allergy andAsthma Proceedings, Volume 27, November-December 2006, pages 451-455).Thus, a Th1/Th2 imbalance caused by over-response of Th2 inducesdiseases such as atopic dermatitis. In addition, as described above,over-response or insufficient response of Th1 or Th2 causes an outbreakof diseases. If the Th1 response decreases and the Th2 responseincreases, cancers, atopic diseases, allergies, and autoimmune diseasesare known to be caused (Elenkov and Chrousos, Stress hormones, Th1/Th2patterns, pro/anti-inflammatory cytokines and susceptibility to disease,Trends in Endocrinology and Metabolism, Volume 10, November 1999, pages359-368). Thus, it is expected that Lactobacillus plantarum CJLP243 maybe applied not only to atopic diseases and allergies but also to cancersand autoimmune diseases since Lactobacillus plantarum CJLP243 controls aTh1/Th2 imbalance by promoting Th1-type reactions.

Another aspect of the present invention provides a composition forenhancing immune response, comprising Lactobacillus plantarum CJLP243.The composition for enhancing immune response is effective forreinforcing immune response since Lactobacillus plantarum CJLP243 is alactic acid bacterium that is effective for enhancing immune response asdescribed above. In particular, as proven by the examples describedbelow, Lactobacillus plantarum CJLP243 is effective for activatinggrowth of general immune cells, and thus the composition for enhancingimmune response is effective for preventing or treating digestive tract(intestinal) infection, urogenital infection, respiratory infection,helicobacter infection and allergic reaction. Furthermore, thecomposition for enhancing immune response is effective for preventing ortreating the diseases caused by a Th1/Th2 imbalance, since CJLP243 iseffective for promoting Th1 response. Thus, the composition forenhancing immune response may be effectively used for preventing ortreating atopic disease, allergies, cancer and autoimmune disease. Theautoimmune diseases include asthma, hay fever, and the like.

The composition for enhancing immune response may be orallyadministered, but the method of administering the composition is notlimited thereto. The dose may vary according to the type of disease,immunity to which need to be enhanced, the degree of seriousness, age,gender, and ethnicity of patients, and purposes of the treatment orprevention. In general, 10 million to 100 billion bacteria may beadministered to an adult.

The composition for preventing or treating intestinal diseases,comprising Lactobacillus plantarum CJLP243 and the composition forenhancing immune response comprising Lactobacillus plantarum CJLP243,use lactic acid bacteria whose safety is proven, and thus thecompositions may be applied to pharmaceuticals, functional food,cosmetics, livestock feed, or additives to livestock feed, without anyconcern about side effects.

If the composition is used as pharmaceuticals, the composition may beformulated in pharmaceutical formulations that are commonly used in theart. The pharmaceuticals may be formulations for oral administrationsuch as liquids, suspensions, powder, granules, tablets, capsules,pills, or extracts.

While the composition is formulated, pharmaceutically acceptablecompatible excipients or additives may be added to the formulations. Theformulation for oral administration may include at least one selectedfrom the group consisting of a diluent, a lubricant, a binder, adisintegrant, a sweetner, a stabilizer, and a preservative, as theexcipient, and at least one selected from the group consisting of aflavoring agent, a vitamin, and an antioxidant, as an additive.

The excipient and the additive may be any pharmaceutically acceptablematerial. In particular, the diluent may be lactic acid, corn starch,soybean oil, microcrystalline cellulose, or mannitol, the lubricant maybe magnesium stearate or talc, and the binder may be polyvinylpyrrolidone or hydroxypropylcellulose. In addition, the disintegrantsmay be calcium carboxymethylcellulose, sodium starch glycolate,polacrilin potassium, or crospovidone, the sweetner may be white sugar,fructose, sorbitol, or aspartame, the stabilizer may be sodiumcarboxymethylcellulose, β-cyclodextrin, white wax, or xanthan gum, andthe preservative may be methyl paraoxybenzoate, propyl paraoxybenzoate,or potassium sorbate.

In addition to the above substances, a natural flavor such as plumflavor, lemon flavor, pineapple flavor, or herb flavor, a natural fruitjuice, a natural colorant such as chlorophylin or flavonoid, asweetening agent such as fructose, honey, sugar alcohol, or sugar, or anacidifier such as citric acid or sodium citrate, or combinations thereofmay be added to the formulation in order to improve taste.

This method of formulation, and excipients and additives for theformulation are described in detail in Remington's PharmaceuticalSciences (19th ed., 1995).

The composition may be used as food. The food may include not onlyfunctional food but also everyday food. The composition used asfunctional food may be formulated into a variety of formulations thatare commonly used in the art with sitologically acceptable excipients oradditives. The functional food may be powder, granules, tablets,capsules, suspensions, emulsions, syrups, liquids, extracts, tea, jelly,drinks, or the like. Any sitologically acceptable excipients oradditives that are commonly used in the art may be used.

Due to its effectiveness for preventing or treating atopic diseases, thecomposition may be used in cosmetics. The composition used in cosmeticsmay be formulated in a variety of formulations that are commonly used inthe art. During the preparation of formulations, excipients or additivesthat are acceptable for cosmetics may be added thereto.

The composition may be used as livestock feed or livestock feedadditives.

If the composition is used as a livestock feed additive, the compositionmay be a liquid with high concentration ranging from 20 to 90% or may beprepared as powder or granules. The livestock feed additive may includeat least one selected from the group consisting of an organic acid suchas citric acid, fumaric acid, adipic acid, lactic acid, or malic acid,phosphate salt such as sodium phosphate, potassium phosphate, acidicpyrrophosphate, or polyphosphate (polymerized phosphate), and a naturalantioxidant such as polyphenol, catechin, α-tocopherol, rosemaryextract, vitamin C, green tea extract, licorice extract, chitosan,tannic acid, or phytic acid. The composition used as livestock feed maybe formulated in a variety of formulations that are commonly used in theart with ingredients commonly used in livestock feed.

The livestock feed additive and livestock feed may include grains suchas powdered or pulverized wheat, oats, barley, corn, or rice; plantprotein livestock feed containing rape, bean, or sunflower as a mainingredient; animal protein livestock feed such as blood powder, meatpowder, bone powder, or fish powder; sugar; and dairy products such aspowdered milk and whey powder. The livestock feed additive and livestockfeed may further include nutrient supplements, digestion and absorptionassisting agents, growth promoting substances, or the like.

The livestock feed additive alone may be administered to animals or maybe combined with another livestock feed additive in edible excipients tobe administered. In addition, the livestock feed additive may beadministered as a top dressing to the livestock feed or as a mixturewith the livestock feed. Alternatively, the livestock feed additive maybe orally administered separately from the livestock feed as a separateformulation. If the livestock feed additive is administered separatelyfrom the livestock feed, it is combined with pharmaceutically acceptableedible excipients to prepare an immediate release or sustained releaseformulation. The edible supports may be solid or liquid, such as cornstarch, lactose, sucrose, bean flake, peanut oil, olive oil, sesame oil,or propylene glycol. If a solid excipient is used, the livestock feedadditive may be in the form of tablets, capsules, powder, troches orlozenges, or a non-dispersed top dressing. If a liquid excipient isused, the livestock feed additive may be in the form of gelatin softcapsules, a syrup suspension, an emulsion, or a solution.

The livestock feed may include organic cereal flour containing proteinthat is commonly used to satisfy the dietary demand of animals. Theprotein-containing cereal flour may include corn, bean flour, orcorn/bean flour mix.

In addition, the livestock feed additive and livestock feed may includeadditives such as a preservative, a stabilizer, a wetting agent, anemulsifier, and a solubilizing agent. The livestock feed additive may beadded to the livestock feed by infiltration, spraying, and mixing.

The livestock feed or livestock feed additive may be applied tolivestock feeds for various animals such as mammals, poultry, and fish.The mammals include pigs, cows, sheep, goats, rodents forexperimentation, and pets (e.g., dogs and cats). The poultry includeschicken, turkeys, ducks, geese, pheasants, and quails, and the fishincludes trout. However, the livestock is not limited thereto.

Advantageous Effects of Invention

As described above, Lactobacillus plantarum CJLP243 according to thepresent invention is a probiotic having excellent acid resistance, bileresistance, and strong adhesion to intestinal epithelial cells, and thusis effective for relieving intestinal disorders. Furthermore,Lactobacillus plantarum CJLP243 is effective for preventing or treatingvarious diseases due to its immune-enhancing effects resulting frompromoting the growth of general immune cells including T cells.Particularly, Lactobacillus plantarum CJLP243 promotes Th1 response andthus it is effective for treating diseases caused by a Th1/Th2 imbalancedue to over-response of Th2. Therefore, Lactobacillus plantarum CJLP243according to the present invention may be applied to a composition fortreating intestinal diseases and a composition for enhancing immuneresponse.

BRIEF DESCRIPTION OF DRAWINGS

The above and other features and advantages of the present inventionwill become more apparent by describing in detail exemplary embodimentsthereof with reference to the attached drawings in which:

FIG. 1 is a graph illustrating acid resistance of Lactobacillusplantarum CJLP243;

FIG. 2 is a graph illustrating bile resistance of Lactobacillusplantarum CJLP243;

FIG. 3 is a graph illustrating an ability of Lactobacillus plantarumCJLP243 to adhere to intestinal epithelial cells.

FIG. 4 is a graph illustrating the results of a MTT assay which wasconducted on immune cells from a spleen, which were isolated from miceand cultured ex vivo, and then treated with Lactobacillus plantarumCJLP243, in comparison with main mitogen treatment groups, a β-glucantreatment group and a Lactobacillus rhamnosus GG treatment group.

FIG. 5 is a graph illustrating the results of an IFN-γ assay, which wasconducted on immune cells from a spleen which were isolated from miceand cultured ex vivo, and then treated with Lactobacillus plantarumCJLP243, in comparison with a main mitogen treatment group, a β-glucantreatment group and a Lactobacillus rhamnosus GG treatment group.

FIG. 6 is a graph illustrating a body weight measured just beforekilling mice orally administered with Lactobacillus plantarum CJLP243strains, in comparison with a negative control group and a Lactobacillusrhamnosus GG administration group.

FIG. 7 is a graph illustrating results obtained by measuring a change inT cell population in order to identify the effect of Lactobacillusplantarum CJLP243 strains on immune cells in the spleen isolated afterkilling the mice orally administered with Lactobacillus plantarumCJLP243 strains, in comparison with a negative control group and aLactobacillus rhamnosus GG administration group.

FIG. 8 is a graph illustrating results obtained by measuring a change inCD4 T cell population in order to identify the effect of Lactobacillusplantarum CJLP243 strains on the immune cells in the spleen isolatedafter killing the mice orally administered with Lactobacillus plantarumCJLP243 strains, in comparison with a negative control group and aLactobacillus rhamnosus GG administration group.

FIG. 9 is a graph illustrating results obtained by measuring a change inCD8 T cell population in order to identify the effect of Lactobacillusplantarum CJLP243 strains on the immune cells in the spleen isolatedafter killing the mice orally administered with Lactobacillus plantarumCJLP243 strains, in comparison with a negative control group and aLactobacillus rhamnosus GG administration group.

FIG. 10 is a graph illustrating results obtained by measuring the amountof IL-2 produced by T cells from spleen cells which were obtained fromthe spleen isolated after killing the mice orally administered withLactobacillus plantarum CJLP243 strains and then stimulated with ConA exvivo, using an enzyme-linked immunosorbent assay (ELISA) method, inorder to identify the effect of Lactobacillus plantarum CJLP243 on theactivation of T cells, in comparison with a negative control group and aLactobacillus rhamnosus GG administration group.

FIG. 11 is a graph illustrating the results obtained by measuring theamount of IFN-γ produced by T cells from spleen cells which wereobtained from the spleen isolated after killing the mice orallyadministered with Lactobacillus plantarum CJLP243 strains and thenstimulated with ConA ex vivo, using the ELISA method, in order toidentify the effect of Lactobacillus plantarum CJLP243 on theactiviatoin of T cells, in comparison with a negative control group anda Lactobacillus rhamnosus GG administration group.

MODE FOR THE INVENTION

Hereinafter, the present invention will be described in detail withreference to the following examples. However, these examples are notintended to limit the purpose and scope of the invention.

Example 1: Isolation and Identification of Lactobacillus plantarumCJLP243 Strains

Lactobacillus plantarum CJLP243 strains isolated from kimchi weresmeared onto a solid MRS medium (Difco, USA) containing 1.5% agar, andincubated at 37° C. for 24 hours. Colonies that were proven to be purewere collected using a loop and incubated in a liquid MRS medium (Difco,USA) at 37° C. for 18 to 24 hours.

Then, morphological and physiological properties of Lactobacillusplantarum CJLP243 strains were measured using a method reported by Kimet. al. (Kim et. al., Leuconostoc inhae sp. nov., a lactic acidbacterium isolated from kimchi, International Journal of Systematic andEvolutional Microbiology, Volume 53, July 2003, pages 1123-1126), andusing API5OCH and API5OCHL kits (Biomerio). The identified morphologicaland physiological properties of Lactobacillus plantarum CJLP243 arelisted in Table 1 above.

In addition, a base sequence of a 16S rRNA gene was analyzed to identifyand classify lactic acid bacteria. The determination and assay of thebase sequence of the 16S rRNA gene were conducted using a methodreported by Kim et. al.(Kim et. al., Leuconostoc kimchii sp. nov., a newspecies from kimchi. International Journal of Systematic and EvolutionalMicrobiology, Volume 50, September 2000, pages 1915-1919. As a result,the base sequence of the 16S rRNA gene of Lactobacillus plantarumCJLP243 is shown at SEQ ID NO: 1 in the sequence list attached hereto.

As a result of a 16S rRNA base sequence assay, a Lactobacillus plantarumCJLP243 strain had the highest homology (99.9%) with a Lactobacillusplantarum standard strain (Lactobacillus plantarum NBRC 15891 ^(T),GenBank accession number AB326351), and was identified, namedLactobacillus plantarum CJLP243, and deposited in the Korean CultureCenter of Microorganisms (KCCM) on Oct. 14, 2009 (Deposition No.:KCCM11045P).

Example 2: Acid Resistance Test in Artificial Gastric Juice and BileResistance Test in Artificial Bile for Lactobacillus plantarum CJLP243Strains

An acid resistance test in an artificial gastric juice was conductedusing an artificial gastric juice prepared using a modified methodreported by Kobayashi et. al., (Kobayashi et. al., Studies on biologicalcharacteristics of Lactobacillus: II. Tolerance of the multipleantibiotic resistance strain, L. casei PSR3002, to artificial digestivefluids. Japan Journal of Microbiology. Volume 29, July 1974, pages691-697). In particular, the artificial gastric juice was prepared byadjusting the pH of a liquid MRS medium to 2.5 using 1N HCl, addingpepsin to a concentration of 1000 unit/ml, and sterilizing the medium.

The Lactobacillus plantarum CJLP243 strains isolated and identified inExample 1 and incubated in a liquid MRS medium at 37° C. for 18 hourswere centrifuged to precipitate the strains, and washed twice withsterilized saline (0.85% NaCl). Then, the strain suspension wasinoculated onto a control medium and the artificial gastric juice to aconcentration of about 10⁷ cfu/ml. The number of surviving strains wasmeasured at the beginning of the inoculation and after 3 hours of theinoculation while culturing at 37° C., in which the total number ofstrains was measured by diluting the strains 10 times with aphosphate-buffered solution (pH 6.8) containing KH₂PO4, Na₂HPO,L-cysteine, HCl, Tween 80, and the like.

A bile resistance test in artificial bile was conducted according to amethod reported by Casey et. al. (Casey et. al., Isolation andcharacterization of anti-Salmonella lactic acid bacteria from theporcine gastrointestinal tract, Letters in Applied Microbiology. Volume39, 2004, pages 431-438). The Lactobacillus plantarum CJLP243 wasincubated in the medium which was prepared by adding 0.3% bile of a bullto the liquid MRS medium used in the acid resistance test above. Thestrains were inoculated in the same manner as in the acid resistancetest above, and the number of surviving strains was measured at thebeginning of the inoculation and after 12 and 24 hours of theinoculation.

The acid resistance test and the bile resistance test of Lactobacillusrhamnosus GG (KCTC 5033) were conducted in the same manner as in thetests of Lactobacillus plantarum CJLP243 for comparison.

The results are shown in FIGS. 1 and 2. FIG. 1 is a graph illustratingacid resistance of Lactobacillus plantarum CJLP243. FIG. 2 is a graphillustrating bile resistance of Lactobacillus plantarum CJLP243.

According to FIGS. 1 and 2, Lactobacillus plantarum CJLP243 had equal orgreater acid resistance and bile resistance compared to other lacticacid bacteria. This indicates that Lactobacillus plantarum CJLP243according to the present invention may reach intestines without beingaffected by gastric juice and inhabit the intestines without beingaffected by bile.

Example 3: Adhesion of Lactobacillus plantarum CJLP243 Strains toIntestinal Epithelial Cells

HT-29 was obtained from Korean Cell Line Bank (KCLB) as an animal cellto test adhesion to intestinal epithelial cells, and the test wasconducted using methods reported by Kim et. al. (Kim et. al., Probioticproperties of Lactobacillus and Bifidobacterium strains isolated fromporcine gastrointestinal tract, Applied Microbiology and Biotechnology,Volume 74, April 2007, pages 1103-1111) and by Hirano et. ai.(Hirano et.al., The effect of Lactobacillus rhamnosus on enterohemorrhagicEscherichia coli infection of human intestinal cells in vitro,Microbiology and Immunology, Volume 47, 2003, pages 405-109).

HT-29 cells were cultured in a RPMI 1640 (Gibco, USA) medium containingheat inactivated 10% fetal bovine serum (FBS), 1% L-glutamine,penicillin G (100 IU/mL), and streptomycin (100 mg/mL) in the presenceof 5% CO₂ at 37° C. In order to measure an adhesion ability and anadhesion inhibitory ability, HT-29 cells were divided into a 24-wellplate such that the number of HT-29 cells per well was 1.0×10⁵ cell/mL.The HT-29 cells were cultured while altering the culture media everyother day until a monolayer was completely formed. The completely formedmonolayer of HT-29 cells was washed five times with PBS buffer solutionat 25° C., and 0.5 mL of a RPMI 1640 medium without antibiotics wasadded thereto.

Lactobacillus plantarum CJLP243 was suspended in a RPMI medium to aconcentration of about 1.0×10⁹ cfu/mL, and the suspension was inoculatedinto the 24-well plate prepared above and incubated in the presence of5% CO₂ at 37° C. for 2 hours. When the incubation was completed, the24-well plate was washed three times with the PBS buffer solution whilestirring at 200 rpm for 3 minutes in order to remove strains that didnot adhere to the well-plate and to identify strains that have anability to adhere to the cell over the washing. After the washing, 0.2%trypsin-EDTA was added into the wells to isolate the adhered cells. Theisolated cells were diluted in peptone water by way of a serial dilutionmethod and smeared on a MRS-agar plate, and then cultured at 37° C. for24 hours. After the culturing, the number of strains was counted.

Separately, in order to identify partial adhesion, after a cover glasscompletely sterilized by immersing it in 70% alcohol for one day wasattached to the bottom of a petri-dish and HT-29 cells were incubated,the same amount of lactic acid bacteria as used above was added thereto,and then cultured and washed in the same manner as described above.Lactic acid bacteria that were not washed and adhered to the HT-29 cellswere dried and stained using Gram staining. The stained bacteria wereobserved using an optical microscope and the number of strains wasmeasured. The same experiments were conducted using Lactobaillusrhamnosus GG (KCTC 5033).

The results are shown in FIG. 3. FIG. 3 is a graph illustrating anadhesion ability of Lactobacillus plantarum CJLP243 to intestinalepithelial cells.

Referring to FIG. 3, Lactobacillus plantarum CJLP243 had a greateradhesion ability to intestinal epithelial cells after 24 hours comparedto Lactobacillus rhamnosus GG (KCTC 5033) commercially well known as aprobiotic. According to these results, it can be seen that theLactobacillus plantarum CJLP243 according to one aspect of the presentinvention is capable of adhering to intestinal epithelial cells, therebyimproving intestinal environments.

Example 4: Safety Test of Lactobacillus plantarum CJLP243 Strains

In order to evaluate safety of the strains isolated in Example 1, ahemolysis test, a gelatin liquefaction test, a hazardous metabolite(ammonia) formation test, and a phenylalanine deaminase test wereconducted according to safety test methods suggested by a collectivestandard of the Korean Bio Venture Association.

The results are shown in Table 2 below.

TABLE 2 Lactobacillus plantarum Safety of CJLP243 Test gelatinphenylalanine ammonia liquefaction deaminase hemolysis formation Strainstest test test test CJLP243 negative negative α-hemolysis, negative safe

According to the results, Lactobacillus plantarum CJLP243 was negativefor the gelatin liquefaction test, the hazardous metabolite (ammonia)formation test, and the phenylalanine deaminase test, and showedα-hemolysis which is estimated not to be related to a pathogen. Thus, itwas proven that Lactobacillus plantarum CJLP243 can be safelyadministered to the human body.

Example 5: The Growth of Mouse Immune Cell by way of Ex Vivo Experiment

In order to identify the immune enhancing ability of Lactobacillusplantarum CJLP 243, the growth of mouse immune cells was identified byan MTT assay. The MTT assay was conducted as follows according to themanufacturer's instructions of an MTT assay kit (promega, cat numberG5430). First, immune cells obtained by eliminating blood cells fromsplenocytes of a B6 mouse were diluted in a RPMI 1640 medium containing10% fetal bovine serum. Afterwards, the immune cells were seeded intothe 96-well plate in the number of 1×10⁶ cells/well (total volume 200

). After the seeding of the immune cells was completed, liveLactobacillus plantarum CJLP 243 (5×10⁶ cells) were added to each welland cultured at 37° C. in a 5% CO₂ incubator for 3 days. After 3 days,100

of medium was removed 1 hour before the MTT assay in order to preparefor the MTT assay. Then, 20

of MTT solution (2 mg/ml) was dispensed into each well, and thencultured at 37° C. for 1 hour. Afterwards, absorbance was measured at490 nm using an ELISA reader (Molecular device, USA). This procedure wasrepeated at least 3 times. In this procedure, positive control groups,each at a concentration of 10 μg/ml, used concanavalin A (con A; sigmacat number L6397), phytohemaglutamine (PHA; sigma cat number L8902), orlipopolysaccharide (LPS; sigma cat number L4516) which are mainmitogens. Furthermore, 99% pure β-glucan was purchased from SigmaChemical (cat number G5011, USA) and was used in the final concentrationof 1.25 μg/ml to compare the immune enhancing ability. The results areshown at FIG. 4.

FIG. 4 is a graph illustrating the results of the MTT assay which wasconducted on immune cells from a spleen, which were isolated from amouse and cultured ex vivo, and then treated with Lactobacillusplantarum CJLP243.

Referring to FIG. 4, it can be said that Lactobacillus plantarum CJLP243has superior ability to activate splenocyte growth compared to the mainmitogens, β-glucan, and Lactobacillus rhamnosus GG (KCTC 5033), and thusis confirmed to be able to induce the enhanced immunity.

Example 6: Ability of Producing IFN-γ using Cytokine Assay

In order to identify an immune enhancing ability of Lactobacillusplantarum CJLP243 on a foreign antigen, the production ability of IFN-γ,a Th1-type cytokine, was measured using an ELISA assay. First, immunecells obtained by eliminating blood cells from splenocytes of a B6 mousewere diluted in a RPMI 1640 medium containing 10% fetal bovine serum.Afterwards, the immune cells were seeded into the 96-well plate in thenumber of 1×10⁶ cells/well (total volume 200

). After the seeding of the immune cells was completed, liveLactobacillus plantarum CJLP 243 (5×10⁵ cells) were added to each welland cultured at 37° C. in a 5% CO₂ incubator for 3 days. After 3 days,50

of the culture medium was removed and the amount of IFN-γ containedtherein was measured using the ELISA assay. In order to conduct theELISA assay, an ELISA plate was coated with an IFN-γ antibody at roomtemperature for 4 hours, and then the culture medium 50

were added thereto and cultured at room temperature. Afterwards, theculture medium was eliminated from the ELISA plate, which was thencoated with a biotinylated IFN-γantibody (BD Bioscience, cat number554410), which is a second antibody, at room temperature for about 2hours. Color reaction was induced by streptavidine-conjugatedhorseradish peroxidase (vectore, cat number SA-5004) and its substrate,TMB (3,3′, 5,5′-tetramethylbenzidine, KPL, cat number 50-76-03). Afterthe color reaction was conducted, 100 μg/ml of stop solution (3 M HCl)was added to the plate in order to stop the color reaction. At thistime, the color reaction showed the color change from blue to yellow,and the absorbance was measured at 450 nm using an ELISA reader(Molecular device, USA) after the color reaction stopped. This procedurewas repeated at least 3 times. In these procedures, positive controlgroups used concanavalin A (con A; sigma cat number L6397),phytohemaglutamine (PHA; sigma cat number L8902), or lipopolysaccharide(LPS; sigma cat number L4516) which are main mitogens, each in theconcentration of 10 μg/ml. Further, 99% pure β-glucan was purchased fromSigma Chemical (cat number G5011, USA) and used in an amount of 1.25 mgin the final to compare the immune enhancing ability. The results areshown at FIG. 5.

FIG. 5 is a graph illustrating the results of the IFN-γ assay which wasconducted on the immune cells from the spleen which were isolated fromthe mouse and cultured ex vivo, and then treated with Lactobacillusplantarum CJLP243.

Referring to FIG. 5, it can be said that Lactobacillus plantarum CJLP243has inferior ability to the main mitogens that are positive controlgroups, but significantly superior ability to Lactobacillus rhamnosus GG(KCTC 5033) with respect to the IFN-γ production, and thus is confirmedto be able to induce the enhanced Th1-type immunity.

Example 7: Enhanced Immunity of Lactobacillus plantarum CJLP243 Strainwhen it is Orally Administered

A test for identifying the enhanced immunity of a Lactobacillusplantarum CJLP243 strain when it is orally administered was conducted asfollows.

4-week-old female Balb/c mice were purchased and maintained forstabilization for 1 week in an animal breeding room, which was in theSPF condition maintained at a temperature of 24=2° C. and a humidity of55±15%. The mice were provided with general powdered livestock feed towhich some antibiotics were added, and were provided with water adlibitum. Each Lactobacillus rhamnosus GG and Lactobacillus plantarumCJLP 243 was processed in the form of lyophilized powder, which was keptrefrigerated. Lactic acid bacteria was uniformly mixed with powderedlivestock feed and orally administered to the mice at a dose of 2.5×10¹⁰CFU/day/mouse. The weight of each mouse was measured for 8 weeks duringthe administration, and then all the mice were killed for the followingexperiments.

The spleen was removed from each killed mouse, and its length and weightwere measured. The spleen was smashed using a plunger of a syringe andfiltered through a mesh in order to separate cells, and these processeswere conducted in an ACK buffer. The separated cells were counted usinga microscope.

In order to identify the composition change of immune cells in theseparated splenocytes, T cells in separated cells were stained withanti-Thy1.2-FITC antibodies, and B cells were stained withanti-CD19-FITC antibodies. CD T cells were stained with anti-Thy1.2-FITCantibodies and anti-CD4-PE antibodies, and CD8 cells were stained withanti-Thy1.2-FITC antibodies and anti-CD8-PE antibodies. Stained cells ineach tissue were analyzed for their composition ratio using a FACScan(BD Biosciences, USA).

The separated splenocytes were treated with ConA at the concentration of5 μg/mL to stimulate T lymphocytes. After 24 hours, the supernatant wasremoved and measured for the concentrations of IFN-γ and IL-2, Tlymphocyte cytokines, using an ELISA. A microplate was coated with IFN-γand IL-2 capture antibodies at 4° C. overnight, and then washed with PBS(PBST) containing 0.05% Tween20 and blocked with PBS containing 3% fetalbovine serum. After 1 hour, the microplate was washed and thesupernatant and a standard solution (a standard solution provided by BDBioscience) were added to each well and reacted at 4° C. overnight.Then, the microplate was washed and biotinylated antibodies were addedto each well, and the reaction was conducted at room temperature for 45minutes. Afterwards, the microplate was washed andstreptavidine-alkaline phosphatase was added to each well, and culturingwas conducted at room temperature for 30 minutes. After washing themicroplate, a color reaction was conducted by adding a p-nitrophenylphosphate (Sigma, USA) solution to each well. Absorbance was measured at405 nm using a microplate reader, and the concentration of cytokines wasmeasured using a standard curve.

The results are shown in FIGS. 6 to 11.

FIG. 6 is a graph illustrating the weight measured just before killingthe mice orally administered with Lactobacillus plantarum CJLP243strains, in comparison with a negative control group and a Lactobacillusrhamnosus GG administration group.

Referring to FIG. 6, a Lactobacillus plantarum CJLP243 strainsadministration group was seen to maintain normal body weight without anyabnormalities due to excessive enhanced immunity, and no abnormalitieswere observed with respect to the weight of the spleen and the number ofcells (data is not shown).

FIG. 7 is a graph illustrating the results obtained by measuring thechange in a T cell population in order to identify the effect ofLactobacillus plantarum CJLP243 strains on the immune cells in thespleen isolated after killing the mice orally administered withLactobacillus plantarum CJLP243 strains, in comparison with a negativecontrol group and a Lactobacillus rhamnosus GG administration group.

FIG. 8 is a graph illustrating the results obtained by measuring thechange in a CD4 T cell population in order to identify the effect ofLactobacillus plantarum CJLP243 strains on the immune cells in thespleen isolated after killing the mice orally administered withLactobacillus plantarum CJLP243 strains, in comparison with a negativecontrol group and a Lactobacillus rhamnosus GG administration group.

FIG. 9 is a graph illustrating the results obtained by measuring thechange in a CD8 T cell population in order to identify the effect ofLactobacillus plantarum CJLP243 strains on the immune cells in thespleen isolated after killing the mice orally administered withLactobacillus plantarum CJLP243 strains, in comparison with a negativecontrol group and a Lactobacillus rhamnosus GG administration group.

Referring to FIGS. 7, 8 and 9, Lactobacillus plantarum CJLP243 strainsare identified to increase the number of T cells, CD4 T cells, and CD8 Tcells, and thus it can be said that Lactobacillus plantarum CJLP243enhances immunity by affecting the activation of T cell growth.

Additionally, in order to identify the effect of Lactobacillus plantarumCJLP243 on the activation of T cells, the amount of IL-2 and IFN-γproduced by T cells from spleen cells which were obtained from thespleen isolated after killing the mice orally administered withLactobacillus plantarum CJLP243 strains and then stimulated with ConA exvivo, were measured using the ELISA method. The obtained results areshown in FIGS. 10 and 11.

FIG. 10 is a graph illustrating the results obtained by measuring theamount of IL-2 produced by T cells from spleen cells which were obtainedfrom the spleen isolated after killing the mice orally administered withLactobacillus plantarum CJLP243 strains and then stimulated with ConA exvivo, using the ELISA method, in order to identify the effect ofLactobacillus plantarum CJLP243 on the activation of T cells, incomparison with a negative control group and a Lactobacillus rhamnosusGG administration group.

FIG. 11 is a graph illustrating the results obtained by measuring theamount of IFN-γ produced by T cells from spleen cells which wereobtained from the spleen isolated after killing the mice orallyadministered with Lactobacillus plantarum CJLP243 strains and thenstimulated with ConA ex vivo, using the ELISA method, in order toidentify the effect of Lactobacillus plantarum CJLP243 on the activationof T cells, in comparison with a negative control group and aLactobacillus rhamnosus GG administration group.

Referring to FIGS. 10 and 11, it can be said that Lactobacillusplantarum CJLP243 strains are identified to increase the production of Tcell cytokines over the negative control group, by which Lactobacillusplantarum CJLP243 may induce the activation of T cells and enhancegeneral immunity.

Example 8: Preparation of Probiotics Powder Comprising Lactobacillusplantarum CJLP243

Lactobacillus plantarum CJLP243 identified in Example 1 weremass-produced and lyophilized to make probiotics to be applied to rawmaterials for pharmaceuticals, food, livestock feed, livestock feedadditives, or cosmetics.

The bacteria were incubated in a liquid MRS medium (Difco) at 37° C. for18 hours while regulating the pH at 6.0 using a 25% NaOH solution, andcentrifuged to obtain strains. The collected strains were lyophilized at−40° C. using 5% dextrin and 10% skim milk as lyophilization protectingagents, and the dried strains were pulverized using a mixer at 37° C. toprepare powder. The powdered live bacteria were mixed with excipients,such as glucose, lactic acid, and skim milk, to adjust the number ofbacteria to a desired level and to store the live bacteria, and packedin a sealed aluminum pouch.

The probiotics prepared according to this method may be applied topharmaceuticals, food, livestock feed, cosmetics, or the like, by beingmixed with grain powder used as a raw material of the livestock feed, bybeing mixed with excipients or additives to form pharmaceuticals, suchas tablets and capsules, or food, and by being mixed with raw materialsto form cosmetics.

While the present invention has been particularly shown and describedwith reference to exemplary embodiments thereof, it will be understoodby those of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the present invention as defined by the following claims.

1. A method for treating an immune disease caused by a Th1/Th2 imbalancein favor of a Th2 shift in a subject, comprising: administering acomposition comprising Lactobacillus plantarum CJLP243 to the subject inneed thereof.
 2. A method for improving a microbial environment in agastrointestinal tract of a subject, comprising: administering acomposition comprising Lactobacillus plantarum CJLP243 to the subject inneed thereof.
 3. A method for treating an immune disease in a subject,comprising: administering a composition comprising Lactobacillusplantarum CJLP243 to the subject in need thereof, wherein the immunedisease is caused by a Th1/Th2 imbalance selected from the groupconsisting of allergic diseases, atopy, cancer, and enteropathy.
 4. Themethod of claim 1, wherein the composition is selected from the groupconsisting of pharmaceuticals, food, animal feed, feed additives andcosmetics.
 5. The method of claim 2, wherein the composition is selectedfrom the group consisting of pharmaceuticals, food, animal feed, feedadditives and cosmetics.
 6. The method of claim 3, wherein thecomposition is selected from the group consisting of pharmaceuticals,food, animal feed, feed additives and cosmetics.
 7. The method of claim1, wherein the subject is a human.
 8. The method of claim 2, wherein thesubject is a human.
 9. The method of claim 3, wherein the subject is ahuman.
 10. The method of claim 1, wherein the composition isadministered orally.
 11. The method of claim 2, wherein the compositionis administered orally.
 12. The method of claim 3, wherein thecomposition is administered orally.